SUMMARY Our goal is to determine the molecular mechanism of soluble guanylate cyclase (sGC) activation by NO via discovery of Long-range Interaction NetworKs of amino acids (LINKs) using a novel activating-mutation reporter screening assay. Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The only effective therapies aim to lower intraocular pressure (IOP) to limit disease progression. However novel molecular targets and therapies are necessary to halt or reverse disease progression and improve patient quality of life. The sGC enzyme, already an established drug target in peripheral vascular disorders, plays a prominent role in the development of POAG. sGC controls the NO-sGC-cGMP pathway by producing cGMP, which plays a key role in IOP regulation. The enzyme is regulated by nitric oxide (NO) binding to its regulator domain transmitting a yet to be characterized activating signal to the catalytic domains that increases cGMP output several hundred fold. Despite the recent determination of a low-resolution structure of full-length sGC, the mechanism by which NO allosterically enhances sGC activity remains unknown, limiting the utility of sGC as a therapeutic target for POAG. Key to elucidating the structural requirements for NO activation of sGC is the mechanism by which the catalytic domains achieve their high-activity conformation. We propose to use a retro-mechanistic approach to first define the high-activity structural transitions in the catalytic domains while subsequently tracing them back to the NO-binding event in the regulatory domain. We hypothesize that LINKS play a critical role in NO- dependent formation of an active site conformation that is optimized for full enzymatic activity. We have already defined parts of these LINKs in our new preliminary data confirming a novel role for residues located in an interfacial ?-flap as key modulators of the orientation-dependent activation of the catalytic domains. Fully defining sGC-activating LINKs will be crucial to (i) understand the structural transitions required for sGC activation and how they are modulated by NO and (ii) design small molecule modulators of sGC that modulate these transitions. Results from our studies will identify new sites (referred to as ?LINKs? in our proposal) that promote transitions between the inactive and active sGC catalytic states. Defining these transitions will be the first step to understand how NO allosterically activates full-length sGC. In the long-term, results from this research will allow us to specifically modulate the NO-sGC-cGMP pathway that plays a key role in regulating IOP, crucial to the etiology of POAG.